Structural Studies of Noroviruses

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PROJECT SUMMARY: Project 3 The major goal of Project 3 is to provide a detailed structural and mechanistic understanding of human norovirus (NoV) evolution and replication to aid in the design and development antiviral strategies. Human NoVs are the leading cause of epidemic acute gastroenteritis. Susceptibility to these viruses is determined by genetically controlled expression of histo-blood group antigens (HBGAs), which are also critical for NoV attachment to host cells. As a result of sequence changes in the P domain of the capsid protein VP1 that harbors the HBGA binding site, these viruses show strain-dependent variability in HBGA specificity presenting a fascinating case study in how genotypic variations allow for exploitation of the polymorphic nature of HBGAs in host populations to counter herd immunity and cause epidemics. Recent studies have shown human antibodies that block HBGA binding correlate with protection from NoV-associated illness (Project 1). Several studies have suggested a correlated interplay between antigenicity and HBGA specificity in driving the evolution of NoVs. The goal of AIM 1 is to provide the structural basis for such interplay by structurally characterizing how HBGA-blocking antibodies interact with NoV strains using currently available human monoclonal antibodies and those newly developed in Project 1, using X-ray crystallography and cryo-EM techniques. While the emphasis in AIM 1 is on virus-host interactions, the emphasis in our following two AIMs is on two key virus encoded proteins that regulate virus replication, both of which are potential targets for small molecule drug discovery. AIM 2 focuses on the viral protease that is critical for polyprotein processing. Based on a recent exciting finding by us and others that NoV protease binds to viral RNA, we have hypothesized that it plays a hitherto uncharacterized role in genome replication. In the first part of AIM 2, our goal is to determine the structural basis of genogroup-dependent substrate interactions and inhibition in order to provide a rational framework for the design and optimization of inhibitors in collaboration with Project 1. In the second part of this aim, our goal is to structurally characterize NoV protease interaction with viral RNA, and in collaboration with Project 2, probe into the role of this interaction in viral replication. AIM 3 will focus on Norwalk virus NTPase, p41, a member of a highly conserved family of proteins encoded by a wide variety of (+)RNA viruses that plays a critical role in virus replication by remodeling cellular membranes into vesicular compartments in infected cells. During the current grant period, we provided the first structural characterization of such a protein from our studies on p41. The goal of this aim is to take the next step of addressing mechanism-related questions using a combination of crystallography, cryo-EM and cell-based techniques and to correlate our observations to the functional aspects of this protein during virus replication in collaboration with Project 2. We expect our proposed studies will provide novel structural and mechanistic insight that will have significant impact on immunological, translational, and replication-related aspects of human NoVs.